C. difficile is a leading cause of antibiotic-associated infection in hospitals worldwide, including such infections as hospital-acquired diarrhea and pseudomembranous colitis. Disease symptoms are caused by homologous toxins A and B (TcdA and TcdB), which form membrane-spanning translocation pores through which associated cytotoxic enzymatic domains are delivered into target cells of the colonic epithelium. This leads to cellular death and tissue damage. Binding to target cells triggers toxin internalization into acidified vesicles, whereupon cryptic segments from within the translocation domain unfurl and insert into the membrane of the epithelial cell, creating a transmembrane passageway to the cytosol. Despite a wealth of information for the enzymatic domains that act once inside the cell, little is known about the translocation pore and its role disease pathogenesis.
TcdA and TcdB are the primary virulence determinants of pathogenic C. difficile. TcdA and TcdB are responsible for the symptoms associated with infection, including diarrhea and pseudomembranous colitis. TcdA and TcdB are large (approximately 308 kDa and 270 kDa, respectively), homologous toxins sharing 48% sequence identity. An amino acid sequence representing TcdB is provided in FIG. 1 (SEQ ID NO:1). TcdA and TcdB appear to intoxicate target cells using a strategy that is similar to a number of smaller A-B toxins, such as anthrax toxin and diphtheria toxin (DT). In addition to a cytotoxic enzymic A-domain and receptor binding B-domain responsible for binding and translocating the A-domain into cells, TcdA and TcdB are equipped with an internal autoprocessing domain that proteolytically cleaves and releases the N-terminal glucosyltransferase domain in response to intracellular inositol hexakisphosphate.
The series of events leading to the delivery of the A-domain into cells begins with toxin binding to an as yet identified receptor on target cells via the C-terminal receptor-binding domain (the B-domain), which triggers toxin internalization into acidified vesicles via clathrin-mediated endocytosis. In the endosome, cryptic regions from within the large ˜1000 amino acid translocation domain emerge and insert into the endosomal membrane, creating a pore that is believed to enable translocation of the N-terminal glucosyltransferase (the A-domain) into the cytosol. Processed and released A-chains enzymatically glucosylate and thereby inactivate intracellular Rho and Ras family GTPases, leading first to cytopathic effects (such as cell rounding), and later cytotoxic effects, such as apoptosis and necrosis.
Like many other A-B toxins that mediate their own delivery into cells, high-resolution structures of the enzymic A-domains and the receptor-binding portion of the B-domains of glucosylating toxin family members are known, while the structure and mechanism of the pore-forming translocation domain remains poorly characterized. These inter-connected processes have been proposed to be mediated by the central ˜1000 amino acid D-domain (at about aa 801-1850). However, absent structural information for this domain in either the pre-pore or pore state, no framework exists for resolving the functional determinants for this large domain that govern pore formation and translocation. It is established that in response to acidic pH, the D-domain undergoes a conformational change that results in the formation of ion-conductive pores in both biological membranes and artificial lipid bilayers.
It has been hypothesized that the cluster of 172 hydrophobic, highly conserved amino acids in the middle of the translocation domain (i.e., residues 958-1130 in TcdA; and, 956 to 1128 in TcdB) comprised some, if not all, of the segments that form the translocation pore. See von Eichel-Streiber et al. (1992), Mol Gen Genet 233(1-2):260-268. Demonstrating this, however, has been challenging, in large part due to difficulties associated with manipulating Clostridial toxin genes at the genetic level. The recent availability of clones of both TcdA and TcdB in Bacillus megaterium expression plasmids, which enable the high-level production of stably folded toxin, has facilitated research in this direction, however studies specifically addressing the structure and function of the translocation domain have been limited to large-fragment deletions to probe function (Genisyuerek et al. (2011) Mol Microbiol 79(6)1643-1654; Zhang et al. (2013) PLoS One 8(3):e58634).
U.S. patent application Ser. No. 13/486,550, filed Jun. 1, 2012 and given US Patent Publication No. US 2012/0276132 A1, describes a recombinant toxin of C. difficile which is based on a 97 amino acid deletion in the transmembrane domain.
There is a need for a vaccine to C. difficile that can target toxic proteins, and that could elicit adequate systemic or mucosal immunity to prevent infection or reduce the severity of infection. It is desirable to provide a protein that is highly similar to the native toxin of C. difficile, in which toxicity is reduced or eliminated.